Phloem is an essential tissue for the long distance transport of photoassimilates. Sieve elements, highly specialized phloem cells, are the primary cells involved in long distance transport. P-proteins (phloem-proteins) are a major component of the cytoplasmic contents of sieve elements. P-proteins are synthesized very early in phloem ontogeny and persist in senescent sieve elements. Anatomical observations, combined with the physical characteristics of P-proteins, have led investigators to suggest that P-proteins may serve as wound sealing mechanism to prevent the loss of assimilates from disrupted sieve elements (Eschrich, W., in: Transport in Plants I. Phloem Transport., M. H. Zimmerman and J. A. Milburn, Eds, pp. 39-56, 1975).
Biochemical characteristics of cucurbit P-proteins.
The phloem of many species within the family Cucurbitaceae is composed of large diameter sieve elements from which a protein-rich exudate can easily be collected. Two very abundant P-proteins in phloem exudates collected from Cucurbita species have been biochemically characterized: PP1 (phloem protein 1), a 96 kDa (M.sub.r 80-136 kDa) protein, and PP2 (phloem protein 2), a 48 kDa dimeric lectin (Beyenbach, et al., Planta 119:113-124, 1974; Kollman, et al., Planta 95:86-94, 1970; Read, et al., Eur. J. Biochem. 134:561-569, 1983). Both are basic proteins (pI 9.6-10.4) that have similar amino acid compositions (rich in Lys, Leu, Gly, Glx, Asx) and are components of phloem filaments in vivo (Beyenbach, et al., supra; Weber, et al., Exp. Cell Res. 87:79-106, 1974).
The PP1 monomers cross-link with one another by covalent disulfide linkages between cysteines, forming soluble polymers (Beyenbach, et al., Planta 119:113-124, 1974Read, et al., Eur. J. Biochem. 134:561-569, 1983; Sabnis, et al., Planta 145:459-466, 1979; Walker, Biochem. Biophys. Acta 257:433-444, 1972). Upon oxidation in vitro, purified PP1 formed distinct filaments and is considered to be the primary structural protein involved in the formation of slime plugs that are seen at sieve plates in electron micrographs of disrupted vascular tissues (Read, et al., Eur. J. Biochem. 134:561-569 , 1983; Walker, Biochem. Biosphys. Acta 257:433-444, 1972; Walker, et al., Ann Bot 35:773-790, 1971). In the absence of thiol reagents, the large PP1 polymers will continue to cross-link forming an insoluble gel (Kleinig, et al., Planta 127:163-170, 1975).
PP2 is a lectin (hemagglutinin) that specifically binds poly(.beta.-1,4-N-acetylglucosamine) or chitin (Allen, Biochem. J. 183:133-137, 1979; Beyenbach, et al., Planta 119:113-124, 1974; Read, et al., Eur. J. Biochem. 134:561-569, 1983; Sabnis, et al., Planta 142:97-101, 1978). The dimer was thought to be composed of two separate subunits, .alpha. (Mr 26,500) and .beta. (Mr 25,000), joined by disulfide linkages between cysteine residues (Read, et al., Eur. J. Biochem. 134:561-569, 1983). Recent studies in our laboratory indicate that PP2 is a homodimer composed of similar subunits that may exhibit anomalous migration in SDS-PAGE. Purified PP2 remains soluble upon exposure to either atmospheric oxygen or oxidizing agents and is a component of phloem filaments due to covalent linkage to PP1 by means of disulfide bridges (Kleinig, et al., Planta 127:163-170, 1975; Read, et al., Eur. J. Biochem. 134:561-569, 1983; Read, et al., Planta 158:119-127, 1983).
P-protein filaments also may contain a third covalently-linked protein (Read, et al., Eur. J. Biochem. 134:561-569, 1983). This 45 kDa basic protein is much less abundant than the other P-proteins, and very little is known about the interactions of this protein with PP1 and PP2. The SDS-PAGE profile of cucurbit phloem exudate also contains 7-10 low molecular weight (lmw) polypeptides (9-20 kDa) that are not considered to be P-proteins (Read, et al., Eur. J. Biochem. 134:561-569, 1983). Recent findings in our laboratory suggest that some of the lmw proteins are coordinately synthesized with the abundant P-proteins.
Cucurbit phloem structure and P-protein accumulation.
Cucurbit phloem is composed of distinct types of phloem that are distinguished by their structure, origin and location in the stem (Crafts, Plant Physiol. 7:183-225, 1932). The Cucurbitaceae is one of several plant families that have bicollateral vascular bundles composed of internal and external phloem (fascicular phloem). A second feature that adds to the complexity of cucurbit phloem anatomy is the existence of extrafascicular phloem, which occurs in strands within the cortex and in arcs bordering both sides of the bundle (Blyth, Origin of primary extraxylary stem fibers in dicotyledons. Univ. Cal. Berkeley Publ. Bot. 30:145-232, 1958; Crafts, Plant Physiol. 7:183-225, 1932). In addition to the primary phloem, secondary phloem within the vascular bundle is derived from a vascular cambium. Long distance transport of assimilates is thought to occur in the sieve elements of the bicollateral vascular bundles and not in the extrafascicular phloem (Evert, et al., Planta 109:193-210, 1973).
P-protein accumulation during sieve element ontogeny in cucurbit stems has been described at the ultrastructural level (Cronshaw, et al., J. Cell. Biol. 38:25-39, 1968). In immature sieve elements, P-protein can be observed in the cytoplasm as small aggregates of fine fibrils that are intermixed with ribosomes, endoplasmic reticulum and dictyosomes.
In general, the P-protein bodies of the fascicular sieve elements disperse, whereas, the P-protein bodies of the extrafascicular sieve elements remain as aggregates. A recent report suggests that the changing environment within the sieve element, especially changes in osmotic potential, could be responsible for the dispersal of P-protein bodies into filamentous P-protein (Kulikova, Soviet Plant Physiol. 39:734-739, 1992).
Cucurbita leaves also have bicollateral vascular bundles. The abaxial phloem matures after the adaxial phloem and appears to be the primary pathway for transport of photoassimilates out of the leaf (Turgeon, et al., Planta 129:265-269, 1976). The adaxial phloem might transport assimilates to the expanding mesophyll tissues during leaf development when the leaf functions as a sink tissue (Turgeon, et al., Planta 129:265-269, 1976). During sieve element ontogeny, P-protein bodies accumulate in both the abaxial and adaxial phloem. In the mature abaxial sieve elements, most of the P-protein is filamentous and dispersed, whereas the P-protein bodies in the mature adaxial sieve elements remain condensed like in the extrafascicular phloem of the stem (Turgeon, et al., Protoplasma 83:217-232, 1975).
Promoters Active in Phloem Tissue
In recent years, transcriptional promoters have been identified that direct gene expression to the phloem. This is not surprising considering the central function of the phloem as the primary mechanism for long-distance transport within plants. However, in many cases gene expression that is directed by these promoters also occurs in other tissues.
Examples of promoters that direct vascular gene expression as part of their developmental program include regulatory sequences from viral (Benfey et al., EMBO J. 9[6] 1685-1696, 1990) and bacterial (Kononowicz et al., Plant Cell 4:17-27, 1992) genes as well as plant genes (Liang et al., Proc. Natl. Acad. Sci. USA 86:9284-9288, 1989); Keller and Baumgartner, Plant Cell 3:1051-1061). Transcriptional regulatory sequences have also been isolated from phloem-limited DNA viruses, such as the rice tungro virus (Bhattacharyya-Pakrasi et al., Plant J. 4[1] 71-79, 1993) and the commelina yellow mottle virus (Medberry et al., Plant Cell 4:185-192, 1992), that direct phloem-specific gene expression. In addition, the transcriptional regulatory elements of plant genes encoding proteins that have phloem-associated functions, such as sucrose synthase (Yang and Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), glutamine synthetase (Edwards et al., Proc. Natl. Acad. Sci. USA 87:3459-3463, 1990), and a phloem-specific isoform of the plasmamembrane H+-ATPase (DeWitt et al., Plant J. 1[1]: 121-128, 1991), have been shown to direct phloem-specific expression of reporter genes in transgenic plants.